The goal of this study is to elucidate the molecular mechanisms by which a regulatory protein, the lac repressor, interacts with its specific DNA site, the lac operator. The lactose operon of E. coli is chosen as a system in which the general principles of regulation and DNA-protein interaction can be most easily studied. Genetic methods will be used to isolate and characterize further specific repressor mutants; (a) with increased affinity for the operator, (b) which specifically overcome degree centigrade mutants which are degree centigrade in the presence of a tight binding (Itb) repressor will also be isolated. The already isolated repressor mutants will be further characterized by in vitro measurements. All I gene mutations leading to Itb repressors will be mapped. The Maxam-Gilbert procedure for DNA sequencing will be used to determine base pair exchanges which lead to degree centigrade mutations and super-operator mutations DNA sequencing will also be used as a procedure to identify amino acid exchanges in tight binding repressors. A possible regulatory role of the "third operator" will be investigated and the effect of DNA denaturants on the complexes formed by various repressor mutants with a number of operators. Our long-range concern is to identify the amino acid residues of lac repressors and the base pair of the lac operator which contribute to the repressor-operator interaction. This would be a first step in deciphering the code by which proteins recognize specific DNA sequences.